Amphibian Embryos Research Articles

Effects of chlorothalonil on development and growth of amphibian embryos and larvae

Chlorothalonil is a broad spectrum fungicide widely used in agricultural and urban environments, yet little is known regarding its effects on amphibians. We examined effects of chlorothalonil on growth, malformations, and mortality in embryos and larvae of Xenopus laevis and Spea multiplicata, and assessed variation in sensitivity among aquatic organisms using a species sensitivity distribution (SSD). Chlorothalonil induced gut malformations in X. laevis embryos and inhibited growth. Tail degeneration was observed in larvae of both species and reduced tail length to total length ratios occurred at environmentally relevant concentrations (5.9 and 11.0 μg/L). The mechanism of tail degeneration is unclear, but alteration in the expression of genes involved in tail resorption is a hypothesized mechanism. Larval amphibians were more sensitive than invertebrates and fish. Based on our results and the range of reported environmental concentrations, chlorothalonil may pose a risk to larval amphibians in certain habitats and scenarios.

Scaling of Dorsal-Ventral Patterning by Embryo Size-Dependent Degradation of Spemann’s Organizer Signals

Spemann's organizer plays a key role in dorsal-ventral (DV) patterning in the amphibian embryo by secreting diffusible proteins such as Chordin, an antagonist to ventralizing bone morphogenetic proteins (BMPs). The DV patterning is so robust that an amphibian embryo with its ventral half surgically removed can develop into a smaller but proportionally patterned larva. Here, we show that this robust patterning depends on facilitated Chordin degradation and requires the expression of the Chordin-proteinase inhibitor Sizzled on the opposite side. Sizzled, which is stable and diffuses widely along the DV axis, stabilizes Chordin and expands its distribution in the ventral direction. This expanded Chordin distribution, in turn, limits BMP-dependent Sizzled production, forming an axis-wide feedback loop for shaping Chordin's activity. Using bisection assays, we demonstrate that Chordin degradation is dynamically controlled by embryo-size-coupled Sizzled accumulation. We propose a scaling model that enables the DV pattern to adjust proportionally to embryonic axis size.

Dual embryonic origin of the hyobranchial apparatus in the Mexican axolotl (Ambystoma mexicanum)

Traditionally, the cartilaginous viscerocranium of vertebrates is considered as neural crest (NC)-derived. Morphological work carried out on amphibian embryos in the first half of the XX century suggested potentially mesodermal origin for some hyobranchial elements. Since then, the embryonic sources of the hyobranchial apparatus in amphibians has not been investigated due to lack of an appropriate long-term labelling system. We performed homotopic transplantations of neural folds along with the majority of cells of the presumptive NC, and/or fragments of the head lateral plate mesoderm (LPM) from transgenic GFP+ into white embryos. In these experiments, the NC-derived GFP+ cells contributed to all hyobranchial elements, except for basibranchial 2, whereas the grafting of GFP+ head mesoderm led to a reverse labelling result. The grafting of only the most ventral part of the head LPM resulted in marking of the basibranchial 2 and the heart myocardium, implying their origin from a common mesodermal region. This is the first evidence of contribution of LPM of the head to cranial elements in any vertebrate. If compared to fish, birds, and mammals, in which all branchial skeletal elements are NC-derived, the axolotl (probably this is true for all amphibians) demonstrates an evolutionary deviation, in which the head LPM replaces NC cells in a hyobranchial element. This implies that cells of different embryonic origin may have the same developmental program, leading to the formation of identical (homologous) elements of the skeleton.

From time series to biological network regulations: an evolutionary approach

In this paper we present a new methodology, based on genetic algorithms and multiple linear regression, for discovering regulation mechanisms responsible for observed time series in biological networks. The modeling framework employed is called Metabolic P systems; they are deterministic and time-discrete dynamical systems proposed as an effective alternative to ordinary differential equations for modeling biochemical systems. Our methodology is here successfully applied to the mitotic oscillator in early amphibian embryos. Starting from the time series of substances involved in this system, we are able to reconstruct an MP system reproducing the observed dynamics, where the regulatory components were discovered by our evolutionary methodology. In particular, genetic algorithms are used as a variable selection technique to identify the best representation of any regulation function in terms of some given primitive functions.

Teaching the developmental process of amphibian embryo using 3D teaching materials:― Focusing on its effect for learners with various spatial abilities

Teaching the developmental process of amphibian embryo using 3D teaching materials:― Focusing on its effect for learners with various spatial abilities

Effects of Heavy Metals on Amphibian Embryos, Tadpoles, and Adults

Amphibian population declines globally. Environmental contamination by heavy metals has been suspected to the one of the reason for distinction of amphibian which has obligate aquatic life style during larval period. Amphibians have been widely accepted as animal model for the study of endocrine disruption in aquatic ecosystem at molecular as well as individual levels. There are increasing need for toxicological data in amphibians at multiple endpoints for management of contamination and development of safety guidelines. Here, we reviewed toxicological information about toxicity of heavy metals (arsenic, cadmium, chromium, copper, lead, mercury, nickel, zinc) on various end-point of amphibian.

External gill motility and striated muscle presence in the embryos of anuran amphibians

Anuran external gills were assessed for motility and striated muscle content in 16 species from seven families. Motility of three kinds was observed. Pulsatory movements related to heart beat rhythm were common. In embryos developing to a late stage before hatching, movements of the whole embryo were frequent, with gills rearranging as a consequence. The only clearly active movement, presumably muscle driven, was ‘gill flicking’, a posterior movement of the entire gill into the body either on one side only, or both together, followed by a return to the normal spread-out position. Some species may actively spread their gills when hanging from the water surface film, but we did not observe this. In some species, active gill movement developed over time, but we were not able to follow all species over such a developmental sequence. The relationship between active motility and muscle content was good in most cases. Observations on late stage embryos of the aromobatid Mannophryne trinitatis are presented for the first time. In one species, we noted spread external gills being used to adhere hatchlings to a surface.

Neural induction and early patterning in vertebrates

In vertebrates, the development of the nervous system is triggered by signals from a powerful 'organizing' region of the early embryo during gastrulation. This phenomenon--neural induction--was originally discovered and given conceptual definition by experimental embryologists working with amphibian embryos. Work on the molecular circuitry underlying neural induction, also in the same model system, demonstrated that elimination of ongoing transforming growth factor-β (TGFβ) signaling in the ectoderm is the hallmark of anterior neural-fate acquisition. This observation is the basis of the 'default' model of neural induction. Endogenous neural inducers are secreted proteins that act to inhibit TGFβ ligands in the dorsal ectoderm. In the ventral ectoderm, where the signaling ligands escape the inhibitors, a non-neural fate is induced. Inhibition of the TGFβ pathway has now been demonstrated to be sufficient to directly induce neural fate in mammalian embryos as well as pluripotent mouse and human embryonic stem cells. Hence the molecular process that delineates neural from non-neural ectoderm is conserved across a broad range of organisms in the evolutionary tree. The availability of embryonic stem cells from mouse, primates, and humans will facilitate further understanding of the role of signaling pathways and their downstream mediators in neural induction in vertebrate embryos.

The teratogenic effects of sediments from the Yangtze Estuary and adjacent bay, China, on frog embryos

In recent decades, the Yangtze (Changjiang) Estuary has suffered great environmental changes due to intensive human activities in the Yangtze River basin. This study assessed the ecotoxicity of sediments from the Yangtze Estuary with the frog embryo teratogenesis assay—Xenopus (FETAX). The results showed that the sediment extracts induced multiple malformations in embryos of Xenopus tropicalis. In the embryos treated with nearshore extracts, abnormal eyes, narrow fins and hypopigmentation were the dominant phenotypes, followed by enlarged proctodeums. In these embryos, the percentages of total malformations were greater than 50 % at five sampling sites and less than 40 % at the other seven sites. However, in the embryos treated with the offshore extracts, elongation of the proctodeums was the dominant phenotype, followed by bent tails. The percentages of total malformations ranged from 25 to 45 %. Comparatively, the embryos treated with extracts from the nearshore zone showed a greater variety of phenotypes than those treated with extracts from the offshore zone. These results suggest that the sediments showed high teratogenicity to the amphibian embryos. The results also indicate that the teratogencity to X. tropicalis embryos is a useful indicator of the pollution of sediments.

A cell state splitter and differentiation wave working-model for embryonic stem cell development and somatic cell epigenetic reprogramming

Cell fate determination and development is a biology question that has yet to be fully answered. During embryogenesis and in vivo stem cell differentiation, cells/tissues deploy epigenetic mechanisms to accomplish differentiation and give rise to the fully developed organism. Although a biochemistry description of cellular genetics and epigenetics is important, additional mechanisms are necessary to completely solve the problem of embryogenesis, especially differentiation and the spatiotemporal coordination of cells/tissues during morphogenesis. The cell state splitter and differentiation wave working-model was initially proposed to explain the homeostatic primary neural induction in amphibian embryos. Here the model is adopted to explain experimental findings on in vitro embryonic stem cell, pluripotency and differentiation. Moreover, since somatic cells can be reverted to a stem-cell-like pluripotent state through the laboratory procedure called epigenetic reprogramming, erection of a cell state splitter could be a key event in their successful reprogramming. Overall, the cell state splitter working-model introduces a bistable cytoskeletal mechanism that partially explains cell fate determination and biological development. It offers an interdisciplinary framework that bridges the gap between molecular epigenetics and embryogenesis.

Genetic comparison of water molds from embryos of amphibians Rana cascadae, Bufo boreas and Pseudacris regilla

Water molds that cause the disease saprolegniasis have been implicated in widespread mortality of amphibian embryos. However, because of the limitations of traditional identification methods, water mold species involved in die-offs or utilized in ecological studies often remain unidentified or identified only as Saprolegnia ferax. Furthermore, water mold taxonomy requires revision, so very distinct organisms may all be called S. ferax. Recent DNA-based studies indicate that the diversity of water molds infecting amphibian embryos is significantly higher than what was previously known, but these studies rely on culture methods, which may be biased towards taxa that grow best under laboratory conditions. In this study, total embryo-associated DNA was extracted from 3 amphibian species in a pond in central Washington, USA. The internal transcribed spacer (ITS) region of DNA was amplified with primers capable of amplifying a broad array of eukaryotic microorgansisms, and was used to construct clone libraries. Individual clones were sequenced and relationships among newly recovered sequences and previously studied taxa were analyzed using phylogenetics. These methods recovered several new taxa in association with amphibian embryos. Samples grouped into 11 distinct phylotypes with ITS sequence differences ranging from 4 to 28%. The water mold communities recovered differed among Rana cascadae, Bufo boreas, and Pseudacris regilla egg masses. Furthermore, the diversity of water molds increased as egg masses aged, and members comprising this diversity changed over time.

Simulation of the effects of microtubules in the cortical rotation of amphibian embryos in normal and zero gravity

This paper reports the results of computer modeling of microtubules that end up in the cortical region of a one-cell amphibian embryo, prior to the first cell division. Microtubules are modeled as initially randomly oriented semi-flexible rods, represented by several lines of point-masses interacting with one another like masses on springs with longitudinal and transverse stiffness. They are also considered to be space-filling rods floating in a viscous fluid (cytoplasm) experiencing drag forces and buoyancy from the fluid under a variable gravity field to test gravitational effects. Their randomly distributed interactions with the surrounding spherical container (the cell membrane) have a statistical nonzero average that creates a torque causing a rotational displacement between the cytoplasm and the rigid cortex. The simulation has been done for zero and normal gravity and it validates the observation that cortical rotation occurs in microgravity as well as on Earth. The speed of rotation depends on gravity, but is still substantial in microgravity.

A mean field Ising model for cortical rotation in amphibian one-cell stage embryos

We propose a new physical mechanism of cortical rotation generation in one-cell embryos of amphibians based on a phase transition in the ensemble of microtubules localized to the cortical region of the cell interior. Microtubules, protein polymers formed from tubulin heterodimers, are highly negatively charged, which results in strong electrostatic interactions over tens of nanometers, even in the presence of counterions that partially screen electrostatic interactions. A simplified model that offers a plausible representation of these effects is based on the Ising Hamiltonian, which has been robustly applied to explain a wide range of order–disorder transitions in physics, chemistry and other sciences. An Ising model phase transition, especially with the supercooperative flow alignment effect of global rotation of the cortex, provides an alternative to models of cortical rotation based on microtubule polymerization or motor molecules. Insofar as there is any reality to the concept that microtubules are involved in consciousness, we propose that cortical rotation in the one-cell embryo is a better place to look for the purported microtubule entanglement or coherence properties than the adult brain.

Growth, development and incidence of deformities in amphibian larvae exposed as embryos to naphthenic acid concentrations detected in the Canadian oil sands region

Naphthenic acids (NA) have been identified as harmful environmental contaminants that influence survival, growth and development of wildlife. Amphibian larvae are particularly susceptible to waterborne contaminants, but little information exists regarding exposure of amphibian embryos or tadpoles to NA. Our results demonstrate that embryos of Lithobates pipiens and Silurana tropicalis exposed to 2–4 mg/l of a commercial NA blend suffer significant reductions (32% and 25%, respectively) in growth and development upon hatching. Increased incidences of deformities were observed in exposed individuals of both species, but were only significant in L. pipiens. Embryos suffered 100% mortality following exposure to 6 mg/l NAs, and narcosis at lower concentrations. LC50 estimates were 4.10 mg/l for 72-h exposure in L. pipiens and 4.13, 3.51, and 2.95 mg/l for 24-, 48-, and 72-h exposure in S. tropicalis. These data suggest that exposure to NAs at environmentally realized concentrations may negatively affect tadpole populations.

Maternal xNorrin, a Canonical Wnt Signaling Agonist and TGF-β Antagonist, Controls Early Neuroectoderm Specification in Xenopus

Author SummaryA key step during early embryogenesis is the generation of neural precursors, which later form the central nervous system. In vertebrates, this process requires proper dorsal–ventral axis specification, and we know that the canonical Wnt and BMP signaling pathways help pattern the dorsal ectoderm. In this study, we examine other factors that are involved in neuroectoderm development in the frog species Xenopus laevis. We find that maternal Xenopus Norrin (xNorrin) is required for canonical Wnt signaling in the dorsal ectoderm, functions upstream of neural inducers, and is required for neural formation. We also find that xNorrin not only activates Wnt signaling, but also inhibits BMP/Nodal-related signaling. In humans, mutations in Norrin cause Norrie disease. Using Norrin mutants identified in patients with Norrie disease, we find that some Norrin mutants fail to inhibit BMP/Nodal-related signaling (specifically, TGF-β) but retain the ability to activate the Wnt pathway, suggesting that loss of TGF-β inhibition may contribute to Norrie disease development.

Ameliorative effects of sodium chloride on acute copper toxicity among Cope's gray tree frog (Hyla chrysoscelis) and green frog (Rana clamitans) embryos

Urban stormwater runoff is composed of a mixture of components, including polycyclic aromatic hydrocarbons, metals, deicing agents, and many others. The fate of these chemicals is often in stormwater detention ponds that are used by amphibians for breeding. Among aquatic organisms, the toxic mechanism for many metals involves interference with active Na(+) and Cl(-) uptake. Addition of cations has been shown to reduce the toxicity of metals among some aquatic organisms through competitive inhibition, but no studies have investigated the interaction between NaCl and Cu among amphibian embryos and larvae. To determine the degree to which NaCl may ameliorate the toxicity of Cu to amphibian embryos and larvae, the authors exposed Hyla chrysoscelis (Cope's gray treefrogs) and Rana (Lithobates) clamitans (green frogs) to seven levels of Cu and NaCl in fully factorial experiments. When exposure was in artificial hard water, Cu was highly toxic to both species (96-h median lethal concentration [LC50] of 44.7 µg/L and 162.6 µg/L for H. chrysoscelis and R. clamitans, respectively). However, approximately 500 mg/L of NaCl eliminated Cu toxicity over the range of Cu concentrations used in the experiments (maximum 150 µg Cu/L for H. chrysoscelis and 325 µg Cu/L for R. clamitans). The current results suggest that NaCl is likely responsible for the toxic effects of NaCl and metal mixtures that might be typical of runoff from road surfaces in northern latitudes.

Effects of the disinfectant Virkon S on early life-stages of the moor frog (Rana arvalis)

Emerging diseases, such as the chytrid fungus Batrachochytrium dendrobatidis, contribute to global population declines of amphibians. Virkon S is one of the most commonly used disinfectants to reduce risk of spreading such pathogens. Virkon S is classified as harmful to aquatic organisms, but until today no negative effects on tadpoles have been reported. We studied the effects of three concentrations of Virkon S on early life-stages (embryos and hatchlings) of the moor frog Rana arvalis. Overall, Virkon S had no significant effects. However, hatching success was highest in the control treatment, suggesting that Virkon S may have weak negative effects on amphibian embryos. We suggest that further studies are needed to assess the negative effect of Virkon S on amphibians, and recommend that Virkon S is used with care and a minimized run-off into natural wetlands.

The Clay Teaching Materials to Understanding of the Initial Development Stages of Amphibian Embryos and Fate-map.

The Clay Teaching Materials to Understanding of the Initial Development Stages of Amphibian Embryos and Fate-map.

Histology of plastic embedded amphibian embryos and larvae

Amphibians including the South African clawed frog Xenopus laevis, its close relative Xenopus tropicalis, and the Mexican axolotl (Ambystoma mexicanum) are important vertebrate models for cell biology, development, and regeneration. For the analysis of embryos and larva with altered gene expression in gain-of-function or loss-of-function studies histology is increasingly important. Here, we discuss plastic or resin embedding of embryos as valuable alternatives to conventional paraffin embedding. For example, microwave-assisted tissue processing, combined with embedding in the glycol methacrylate Technovit 7100, is a fast, simple, and reliable method to obtain state-of-the-art histology with high resolution of cellular details in less than a day. Microwave-processed samples embedded in Epon 812 are also useful for transmission electron microscopy. Finally, Technovit-embedded samples are well suited for serial section analysis of embryos labeled either by whole-mount immunofluorescence, or with tracers such as GFP or fluorescent dextrans. Therefore, plastic embedding offers a versatile alternative to paraffin embedding for routine histology and immunocytochemistry of amphibian embryos.

Internalizing the vegetal cell mass before and during amphibian gastrulation: vegetal rotation and related movements

The movement of the prospective mesoderm and endoderm to the interior of the amphibian embryo starts in the vegetal cell mass well before the onset of overt gastrulation. By an animally directed movement of cells, the vegetal mass constricts its outer part and expands its inner region including the blastocoel floor, in a process of pregastrulation emboly. Further internalization of the vegetal region has been studied in the Xenopus embryo. At the onset of gastrulation, vegetal rotation sets in at the periphery of the vegetal cell mass, first dorsally and then spreading laterally and ventrally. It consists of an intense inward surging of cells due to active cell rearrangements that can be observed in explants of the vegetal cell mass. In its course, the blastocoel floor expands further and becomes apposed to the blastocoel roof. The boundary between apposed floor and roof forms Brachet's cleft. Another effect of vegetal rotation is the downward and inward movement of the mesodermal marginal zone, constituting the first phase of involution. Together, the upward and outward movement of the peripheral vegetal mass and the downward and inward translocation of the marginal zone lead to an apparent rotation of the whole peripheral region of the gastrula. Vegetal rotation continues to contribute to endoderm internalization to near the end of gastrulation.